Generation of a diversified and self-tolerant T cell repertoire constitutes a major foundation of adaptive immunity. T cell development in the thymus results in a continuous generation of CD4 or CD8 T cells expressing alpha/beta T cell receptor (TCR) and a small number of gamma/delta T cells expressing gamma/delta TCR. The fate of each developing T cell is critically dependent on the choice of TCR gene in rearrangement and TCR mediated selection. Most gamma/delta T cells develop early before progenitor cells fully commit to the alpha/beta lineage. T cell progenitors committed to the alpha/beta lineage rarely switch to the gamma/delta lineage even if they fail to produce a functional alpha/beta TCR. This unidirectional development is largely driven and enforced by positive feed back signals from a successfully expressed TCR chain. Thus, TCR based selections serve as critical checkpoints in T cell development. Transcription factors encoded by the structurally and functionally related E2A and HEB genes have been shown to participate in both TCR gene rearrangement and TCR mediated selection. Our own studies of E2A and HEB and their inhibitor Id3 from the previous funding cycles have contributed to the current understanding of this transcriptional network at various stage of T cell development. Analysis of mice carrying targeted mutations has led to two most recent discoveries which are directly relevant to the current proposal. First, our study implicated a regulatory role for E2A and HEB and their inhibitor Id3 in controlling gamma/delta vs. alpha/beta lineage development. Second, conditional inactivation of both E2A and HEB has revealed a necessary role for E2A and HEB in blocking T cell maturation prior to completion of TCR1 gene rearrangement. Based on these novel findings, we propose two specific aims in the current proposal. Aim 1 is to investigate the regulatory mechanism suppressing the gamma/delta lineage fate among cells committed to the alpha/beta lineage. Aim 2 is to investigate the mechanism by which E2A and HEB suppress T cell maturation prior to TCR expression. Outcomes of these studies should directly impact our understanding of the transcriptional network guiding proper lineage differentiation and selection of functional and self-tolerant T cells. Our study in the past funding cycles has demonstrated that genetic perturbation of this transcriptional network directly causes autoimmune disease and dysregulation of immune function. Current proposal should continuously provide insights into the cause of autoimmune disease relevant to humans. Public Health Relevance: The proposed study is a continuation of our fruitful research on function of E2A/HEB in T lymphocyte development for the past 8 years. We have provided crucial genetic basis for how E2A/HEB transcription factors control differentiation, proliferation, and cell death during T cell development. In addition, our study has led to establishment of important animal models for human autoimmune diseases such as Sjogren's syndrome. In the current proposal, we will focus our research on understanding the mechanism by which E2A/HEB coordinate T cell receptor gene rearrangement and T cell selection events. The proposed study should provide both genetic and molecular basis for the production of a diverse yet non-autoreactive T cell population.